Integrated analysis of mRNA and miRNA in human differentiating muscle cells


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Università degli Studi di Padova Dipartimento di Biologia





Direttore della Scuola : Prof. Giuseppe Zanotti

Coordinatore d’indirizzo: Prof. Fiorella Lo Schiavo

Supervisore Prof. Giorgio Valle

Dottorando : Rusha Guha


Table of Contents ACKNOWLEDGEMENTS ... 1 ABSTRACT ... 2 RIASSUNTO ... 4 CHAPTER 1 ... 6 INTRODUCTION ... 6 1.1.SKELETAL MUSCLE ... 7 1.1.1.Structure ... 7 1.1.2.Myogenesis ... 7 1.1.3.Differentiation ... 8

1.1.4. Skeletal muscle contraction ... 9

1.1.5. Skeletal muscle metabolism ... 10


1.2.1.miRNA biogenesis ... 11

1.2.2.mRNA degradation ... 12

1.2.3.Translational activation ... 13





1.7.REFERENCE: ... 16

CHAPTER - 2 ... 21




2.2.1 Cell Culture (Human primary skeletal muscle cells (CHQ5B)): ... 25

2.2.2 RNA extraction from cells: ... 25

2.2.3 RNA quantification and quality assessment ... 27

2.2.3a. RNA quantification: ... 27

2.2.3b. RNA Quality assessment: ... 28

2.2.4 Cell lysate preparation: ... 29

2.2.5 Protein Analysis ... 29

2.2.6 Antibodies: ... 30

2.2.7 RNA Immunoprecipitation (RIP) ... 31

2.2.8 Transcriptome and miRnome sequencing using Applie Biosystem’s SOLiD and Ion proton technology: ... 31


2.2.9 Commercial RNA: ... 32

2.3.RESULTS: ... 32

2.3.1 Human myoblast, myotube and skeletal muscle tissue differential gene expression profile ... 32

2.3.2 miRNA expression profiling of myoblasts, myotubes and skeletal muscle tissue ... 40

2.3.3 Ago2 associated mRNA ... 46

2.4DISCUSSION: ... 48

2.5REFERENCE: ... 51

CHAPTER-3 ... 54




3.2.1 Cell culture ... 57

3.2.2 Transient transfection with oligonucleotides ... 57

3.2.3 RNA extraction and Whole transcriptome sequencing ... 57

3.3RESULTS: ... 57

3.3.1 miRNA expression upon muscle differentiation ... 57

3.3.2 Transient miRNA over expression caused higher number of genes upregulated and less genes down regulated ... 58

3.3.3 Differential gene expression, Pathway and GO analysis for genes affected by each miRNA ... 59

3.3.3a) hsa-miR-139-5p analysis ... 60

3.3.3b) hsa-miR-532-5p analysis ... 61

3.3.3c) hsa-miR-660-5p analysis ... 62

3.3.3d) hsa-miR-92a-3p analysis ... 64

3.3.3e) hsa-miR-206 analysis (Positive control) ... 65

3.3.4 Common effects exerted by all miRNAs ... 67

3.3.5 Comparison of miR down regulated genes with Ago2 enriched genes in myoblasts ... 69

3.4DISCUSSION: ... 71

3.5 REFERENCE ... 73

CHAPTER - 4 ... 75


4.1 INTRODUCTION: ... 77


4.2.1 Cell Culture ... 78

Human primary skeletal muscle cells (CHQ5B): ... 78

4.2.2RNA EXTRACTION ... 78


4.2.2b Poly (A)+ RNA from CHQ5B cells in 3D culture:... 79

4.2.3 RNA quantification, quality assessment and RNA seq ... 79

4.3 RESULT: ... 79

4.3.1 Cell Observation in 3D culture ... 80

4.3.2 Transcriptome analysis of 2-D, 3-D cultivated myotubes and human adult skeletal muscle tissue ... 81

4.3.3 Gene Ontology (GO) and Pathway analysis ... 84

4.3.4 Transcriptome of 3-D compared with 2-D cultured myotubes ... 85

4.4 DISCUSSION ... 89

4.5 REFERENCES: ... 92

CHAPTER 5 ... 95




5.2.1 Cell culture and stretch ... 99

5.2.3 RNA extraction and transcriptome sequencing ... 101

5.2.4 Immunofluorescence ... 102

5.3RESULTS ... 102

5.3.1 Differential gene expression analysis ... 102

5.3.2 GO and pathway analysis ... 104

5.3.3 Myokine genes over expression as an immediate response to stretch ... 105

5.3.4 3h post stretch response analysis ... 106

5.3.5 40h post stretch response analysis ... 109

5.3.6 Stretching propels the process of muscle cell differentiation at transcriptomic level ... 109

5.4 DISCUSSION ... 111

5.5 REFERENCE ... 113








With sincerest of gratitude I would like to thank my supervisor Prof Giorgio Valle for his valuable guidance through my PhD. I also extend my heartfelt thanks to him for being such an enormous support both at professional and personal level. I feel truly grateful and very lucky for having the opportunity to work with him.

I whole heartedly thank Dr. Georgine Faulkner who is a visiting scientist of our lab and who has provided precious inputs to our project with her great experience of working in the field of muscle biology. I have had a chance to refine my practical skills as researcher with her help. I feel so blessed for having worked with Prof Giorgio Valle and Dr. Georgine Faulkner for their endless help and support through the entire thick and thins I have experienced during my stay in Padova and also for extending their care to make my life comfortable while being away from my country.

I would like to thank my colleague and a fellow co PhD student Lisa Marchioretto who has also worked on the muscle project but towards a different goal. It would have been very difficult to settle in a new country which I did not know the language of without her help. I thank her for her kindness and would like to wish her the best for her PhD work. This PhD project would not have been complete without the excellent technical support, so I do thank the entire technical team of our lab. I also thank Nicola Vitulo a bioinformatician of our lab for his contribution towards analyzing the RNA seq data my research project. Working with the team of Prof Giorgio Valle was a great and enriching experience.

Finally I thank my family, my parents my sister for being there like a backbone support, for all their blessings and good wishes which has given me the courage to sail through the journey of PhD.

Last but not the least I would like to thank the program of Erasmus Mundus because of which I got the opportunity to work in the University of Padova.




Muscles are responsible for the movement of body and take up roughly half of the person’s body weight. Skeletal muscles are the only voluntary muscle tissue in human body, being controlled consciously.

Skeletal muscle cells form when many smaller progenitor cells lump themselves together to constitute long, straight, multinucleated fibers. The proliferating muscle cells are called myoblasts. Myoblasts fuse together to form multinucleated non-proliferating cells called myotubes. The transition from proliferative to differentiated state involves a complete shift of the cell’s transcriptome based on a network of regulation at the molecular level. To uncover the intricacies of molecular activities involved in the process of muscle differentiation it is essential to have deeper and through understanding of the transcriptome in its entirety. RNA seq, which is a high through put sequencing technology, gives us the opportunity to reach into the deepest level of the transcriptome. By means of the RNA seq technology I obtained the in-depth view of the transcriptome of human muscle cells from the proliferative to the differentiated stage. The analysis was extended also to small RNAs, to have a full picture of the transcriptome. I performed the analysis with the specific objectives of identifying the expressed genes and finding out differential gene expression, of both mRNA and miRNA. I also investigated the crosstalk between mRNA and miRNA, employing two separate methods : miRNA over expression and Ago2 immunoprecipitation followed by RNA seq.

The over expression experiments were carried out with 5 different miRNAs and in all cases I found more genes turned up than turned down. These results suggest that miRNA might either have a role in mRNA stabilization or it could play a part in a double negative mechanism by inhibiting some negative factor. By comparing the genes down regulated after miRNA over expression with Ago2-enriched genes we have found several candidate genes which are most likely under the down regulatory control of miRNA.

Skeletal muscle has enormous plasticity and can endure a lot of stress. To study this aspect of muscle we performed mechanical stretch of differentiating muscle cells. With RNA seq we got the in-depth view of the transcriptome as a response to stretch. We performed the analysis at two time points after stretch and found that stretch triggered



immune response genes soon after, but enhanced muscle structural-protein genes expression over a prolonged course of time when the immune response takes a back seat. I believe that our thorough transcriptome analysis, including miRNA and mRNA interaction studies during myogenesis, contributes towards the better understanding of the process regulating muscle development.




I muscoli sono responsabili dei movimenti del corpo e costituiscono circa metà del peso di una persona. I muscoli scheletrici sono i soli tessuti muscolari volontari, essendo controllati coscientemente.

Le cellule del muscolo scheletrico si formano quando diverse piccole cellule progenitrici si conglobano tra loro per formare lunghe affusolate fibre multinucleate. Le cellule muscolari proliferanti sono chiamate mioblasti. I mioblasti si fondono tra loro per formare cellule multinucleate e non proliferanti, chiamate miotubi. La transizione dallo stato proliferante a quello differenziato implica un completo cambiamento del trascrittoma cellulare, basato su una rete di regolazione a livello molecolare. Per scoprire il groviglio di attività molecolari implicate nel processo di differenziamento muscolare è essenziale avere una più profonda ed estesa comprensione del transcrittoma nella sua interezza. L'RNA seq è una tecnologia di sequenziamento massivo che ci offre l'opportunità di accedere ai livelli più approfonditi del trascrittoma.

Con la tecnologia dell'RNA seq ho ottenuto una precisa visione del trascrittoma delle cellule muscolari umane, sia allo stadio proliferativo che a quello differenziato. Per avere una visione completa del trascrittoma, l'analisi è stata estesa anche agli small RNA. Ho svolto queste analisi con l'obiettivo specifico di identificare i geni espressi e di evidenziare in particolare quelli differenzialmente espressi, sia per quanto riguarda gli mRNA che i miRNA. Ho anche analizzato il crosstalk tra mRNA e miRNA, impiegando due diversi metodi: sovraespressione dei miRNA e immunoprecipitazione di Ago2, seguita da RNA seq.

Gli esperimenti di sovraespressione sono stati condotti con 5 diversi miRNA e in tutti i casi ho trovato più geni che hanno aumentato il loro livello piuttosto di geni che l'hanno diminuito. Questi risultati suggeriscono che i miRNA potrebbero avere un ruolo nella stabilizzazione degli mRNA, oppure potrebbero avere un ruolo in un doppio meccanismo negativo, inibendo a loro volta fattori negativi. Confrontando i geni che diminuiscono di livello dopo la sovraespressione di miRNA con i geni arricchiti dall'immunoprecipitazione con Ago2, abbiamo trovato diversi geni candidati per essere sotto il controllo inibitorio dei miRNA.

Il muscolo scheletrico ha una grande plasticità e può sopportare un notevole stress. Per studiare questo aspetto del muscolo abbiamo sottoposto le cellule in differenziamento a



stiramento meccanico. Con l'RNA seq abbiamo ottenuto un'approfondita visione del trascrittoma in risposta allo stiramento. Abbiamo effettuato queste analisi a due diversi tempi dopo lo stiramento ed abbiamo trovato che nel periodo immediatamente successivo allo stimolo vengono sovraespressi geni implicati nella risposta immunitaria, mentre successivamente sono attivati i geni codificanti proteine muscolari strutturali, quando allo stesso tempo la risposta immunitaria viene inibita.

Sono convinta che la nostra approfondita analisi del trascrittoma che include l'interazione di mRNA e miRNA durante la miogenesi, possa contribuire ad una maggiore comprensione di processi che regolano lo sviluppo muscolare.



Chapter 1




1.1 Skeletal muscle

Skeletal muscle is one of the most highly organised structures in the biological world, and is primarily involved in the execution of voluntary movement (1). Skeletal muscle is composed of a number of muscle fibre types that differ with respect to their contractile, metabolic and molecular properties (2). The characteristics of a skeletal muscle is its contractile function

1.1.1 – Structure

Skeletal muscle is composed of many individual muscle fibres (3). Each fibre is covered in endomysium and beneath this, resides the cell membrane or sarcolemma. Structurally, each muscle fibre is composed of many protein bundles called myofibrils, which in turn comprise of alternate dark and light staining filaments (3). The sarcomere is composed of the thin (actin) filaments, the thick (mostly myosin) filaments, and the giant filamentous molecule titin. Titin alos known as connectin is the molecular spring that is responsible for the passive elasticity of the muscle. It connects the Z line to the M line in the sarcomere. The thin filaments are anchored in the Z-line, where they are cross-linked by α-actinin. The thick filament is located centrally in the sarcomere and constitutes the sarcomeric band. The myosin heads, interact with actin during activation. In the A-band titin is inextensible due to its strong interaction with the thick filament. The distance from one Z-line to the next is defined as one sarcomere, the smallest integral contractile unit (1).

1.1.2 Myogenesis

The formation of skeletal muscle involves a series of steps in which multipotential mesodermal precursor cells become committed to a muscle cell fate and then proliferate as myoblasts until they encounter an environment lacking mitogens, at which point they exit the cell cycle and differentiate (4). Skeletal, cardiac, and smooth muscle are each derived from mesodermal precursor cells in different regions of the embryo. Although these three different muscle cell types express many of the same muscle-specific genes, each type is unique with respect to the spectrum of muscle genes they express, their morphology, their ability to divide, and their contractile properties. Therefore, if any shared myogenic program exists it must be modified by different regulatory factors to generate the diversity of three muscle cell types.



Adult myogenic cells are derived mainly from muscle satellite cells that are specialized myogenic cells found during late fetal development. In mice the myogenic precursors in the dermomyotome express Pax3, Pax7 and low levels of the myogenic determination factor Myf5. The paired-domain transcription factors Pax3 and Pax7 act upstream of the primary myogenic basic helix–loop–helix (bHLH) transcription factors Myf5 and MyoD, in myogenic specification. When myogenic determination genes MyoD and Myf-5 are activated the muscle precursor cells are committed to become myoblasts and migrate into the adjacent embryonic connective tissue, or mesenchyme and express other muscle specific genes such as myogenin and MRF4, that after a period of proliferation, induce the fusion of myoblasts into multinucleated and highly specialized skeletal muscle cells called myotubes or myofibres (5). A typical myofibre is cylindrical, large (measuring 1-40 mm in length and 10-50 μm) and multinucleated (containing as many as 100 nuclei). Muscle fiber is a cylindrical multinucleate cell composed of numerous myofibrils that contracts when stimulated. Muscle fibres are the basic contractile units of skeletal muscle and are individually surrounded by a layer of connective tissue and grouped into bundles to form skeletal muscle (6, 7).

1.1.3 Differentiation

Skeletal muscle development involves an initial period of myoblast replication followed by a phase where some myoblasts continue to proliferate while other undergoes terminal differentiation. The process of differentiation involves permanent cessation of DNA synthesis, activation of muscle specific gene function and fusion of single cells into multi nucleated muscle fibers (51). Skeletal muscle differentiation is a tightly regulated process that requires the coupling of muscle-specific gene expression with the terminal withdrawal from the cell cycle. The MyoD family of basic helix-loop-helix (bHLH) skeletal muscle specific transcription factors plays a pivotal role in initiating skeletal muscle differentiation.

MyoD and Myf-5 are the most important members of this protein family and are expressed in proliferating and undifferentiated cells, whereas the expression of other bHLH transcription factors such as myogenin and MRF-4 occurs only during differentiation or in adult mature skeletal muscle, respectively. These four myogenic bHLH proteins are known as myogenic regulatory factors (MRFs). The MRFs are able to bind DNA both in the form of homodimers as well as in the form of heterodimers with ubiquitously expressed transcription factors called E proteins. Their binding sites, called E boxes, share the consensus sequence CANNTG. The binding of the bHLH/E protein to



DNA is essential for the activation of the muscle specific differentiation program (8). The differentiation process starts during embryogenesis, when the appropriate environmental stimuli are encountered. In the myotome of the embryo, the MRF’s present in determined myoblasts (MyoD and Myf-5) initiate a cascade of events that leads to the activation of other transcription factors, the MEF2 family, which are necessary for the transcription of myogenin and other skeletal muscle specific genes. Myogenin itself can also activate MEF2, creating a positive regulatory loop that ensures the maintenance of appropriate levels of these proteins in differentiating skeletal muscle (8, 9). Amongst the earliest muscle genes to be expressed in the myotome are desmin, Titin and α-actin. The expression of the myosin heavy chain gene occurs almost a day after the accumulation of the α-actin protein. Committed myoblasts initiate their transformation into differentiated myotubes by first expressing all the major structural proteins. Then, the other muscle genes are activated following a strict temporal regulation during the embryonic, fetal and postnatal development (10). At the same time a system intervenes to allow the cell to exit from the cell cycle thus permitting tissue specific gene expression, cell fusion and the formation of multinucleated myotubes (8). These events involve both muscle specific transcription factors and ubiquitous cell cycle regulatory proteins. In fact MyoD, that represents the major coordinator of skeletal muscle differentiation, is also able to induce the expression of p21, a potent inhibitor of Cyclin-Dependent Kinases (Cdks), thus forces the cell cycle withdrawal (11). This event inhibits the Retinoblatoma protein (pRb) phosphorylation thus promoting its activation and allowing it to sequester the E2F transcription factor, thus blocking cell cycle progression (12). Moreover MyoD and pRb can directly interact with each other (13).

Many other cell cycle regulatory proteins are involved in this complex picture. In fact the p53 tumor suppressor protein is essential in the process of skeletal muscle differentiation, since p53-impaired cells fail to differentiate (14, 15) even though cell cycle withdrawal takes place in a p53-independent manner. Indeed the p53 tumor suppressor is crucial to elevate un-phosphorylated pRb levels to a threshold sufficient to terminally maintain the cell in G0/G1 (17) and to activate together with MyoD the expression of late muscle differentiation markers (18, 19). The last step in the differentiation process is represented by myoblasts fusion. This event correlates with fibronectin secretion onto the extracellular matrix, to which differentiating cells attach using the α5β1 integrin. After that, myoblasts start aligning. This step is mediated by several cell membrane



glycoproteins, including several cadherins and CAMs. Ultimately cell fusion occurs, mainly through the action of a set of metalloproteinases called meltrins and multinucleated differentiated myofibres are formed (20).

1.1.4 Skeletal muscle contraction

Muscle contraction consists of a cyclical interaction between myosin and actin driven by the concomitant hydrolysis of adenosine triphosphate (ATP) (21). Myosin and actin, the components which respectively form the thick and thin filaments were amongst the first proteins to be purified with reference to muscle function (22). The hexametric protein myosin comprises two heavy chains (220 KDa) and four light chains (20-25 KDa), and forms the thick filaments. The terminus of myosin forms a globular head region required for the hydrolysis of ATP and binding of actin. The four light myosin chains are located between the globular head and the carboxy-terminal rod region (1). Thin muscle filaments are comprised of several proteins; however actin (43 KDa) is by far the most abundant constituent. Thick and thin regions of physical overlap form in which globular myosin heads project from the thick filaments to interact with thin actin filaments. ATP hydrolysis mediates a conformational change in the globular myosin heavy chain head region, resulting in an interaction between the globular head and actin further along the filament and inducing a shortening of the muscle fibre.

Multiple isoforms of myosin heavy chain (MHC) exist, which comprise a family of molecular motors able to modulate the speed of skeletal muscle contraction (23). The contractile speed of a particular muscle fibre may therefore be determined, in part, by the isoform of MHC protein which it expresses. The sarcomeric MHC family consists of at least eight known isoforms, each encoded by a distinct gene located in two multigenic regions on two separate chromosomes (24). Six genes are encoded by a 300 – 600Kb segment on human and mouse chromosomes 17 and 11 respectively, in a cluster arrangement in the order MyH3/MyH2, MyH1/MyH 4, MyH 8/ MyH13 (5’ – 3’). The MyH2, MyH1 and MyH4 genes encode the protein isoforms commonly termed MHC IIA, IIX and IIB. Of the eight sarcomeric isoform genes of MHC, four are known to be expressed in adult skeletal muscle: one “slow-twitch” (Type I) muscle associated MHC isoform is encoded by the MyH7β gene and three “fast-twitch” (Types IIA, IIX and IIB) muscle associated isoforms, associated with increasing contractile speed. A combination



of the latter “fast-twitch” isoforms account for over 90% of MHC in adult skeletal muscle (25).

1.1.5 Skeletal muscle metabolism

Muscle contraction is driven by the hydrolysis of adenosine triphosphate (ATP) (21) which may be derived from the metabolism of fatty acids (β-oxidation) or carbohydrates (glycolysis). The β-oxidation of fatty acids takes place in the mitochondria of muscle fibres. Endothelial lipoprotein lipase (LPL) is involved in the transport of fatty acids from the circulatory system into the myocellular compartment (26). Hormone sensitive lipase (HSL) liberates free fatty acids from the intramyocellular lipid (IMCL) pool which are transported into the mitochondrion for β-oxidation by carnitine palmitoyltransferase 1 (CPT-1). The production of ATP via the β-oxidation of fatty acids is an oxygen-dependent process.

Carbohydrates reach the myofibre from the circulatory system and may be stored as glycogen, converted to triglycerides, or metabolised via glycolysis. In contrast to β-oxidation, the metabolism of carbohydrates via glycolysis is an oxygen independent process; however metabolism under these conditions leads to the production of lactate (26).

1.2miRNA and its role in general

A microRNA is a small non-coding RNA molecule (22 nucleotides) found in plants, animals, and some viruses, which functions in transcriptional ans post-transcriptional regulation of gene expression. Encoded by nuclear DNA in plants and animals and by viral DNA in certain viruses whose genome is based on DNA, miRNAs function via base pairing with complementary sequences within mRNA molecules, usually resulting in gene silencing via translational repression or target degradation. The human genome may encode over 1000 miRNAs, which may target about 60% of mammalian genes and are abundant in many human cell types.

1.2.2 miRNA biogenesis

miRNAs are transcribed into long transcripts mainly by RNA polymerase II, although there are also evidences implicating RNA polymerase III in the transcription of some miRNAs (27). Most of them are polyadenylated in its 3’end and capped at its 5’extremity,



like mRNAs. In the canonical pathway (Fig 10) these primary-miRNAs (pri-miRNAs) folds into a stem loop structure that will be further processed by two RNAse III endonuclease, Drosha and Dicer. In the nucleus, the hairpin structure is cleaved from the flanking regions originating the precursor-miRNA (pre-miRNA) that is ≈70 nucleotide long. This first processing step is catalyzed by Drosha that is helped by a cofactor, DGCR8 (DiGeorge syndrome critical region gene 8) (Pasha in Drosophila). This complex is called the Microprocessor. DGCR8 contains two dsRNA-binding domains that directly interact with the stem-loop and with the flanking region, serving as a molecular anchor to Drosha that carries out the cleavage reaction. The cleavage produces highly exact extremities and is highly regulated. The production of pre-miRNAs not always requires the participation of the microprocessor complex. In fact, a rare alternative pathway has been identified initially in fly and nematodes, but also present in mammals (28, 29, 30). This pathway uses the splicing machinery to liberate introns that mimic the features of pre-miRNAs. These structures are called mirtrons. After being spliced they enter the normal miRNA processing pathway.

Pre-miRNAs are then exported to the cytoplasm by Exportin-5 in a Ran-GTP dependent way, where they will be further processed. In the cytoplasm the terminal loop of the pre-miRNA is cleaved originating a mature dsRNA of approximately 22 nucleotides of length. This step is carried out by Dicer. The PAZ domain of Dicer binds to the 3’overhangs of the pre-miRNAs and this binding determines the cleavage site since that Dicer’s catalytic sites are located precisely two helical turns away from the PAZ domain (bound to the pre-miRNA). In this step, Dicer is assisted by the Tar RNA Binding Protein – TRBP (know as Loquacious in Drosophila), another dsRNA binding protein. At the end of this last processing step Argonaute 2 is recruited to the complex Dicer/TRBP leading to the unwinding of the duplex. At this stage one of the strands, the mature miRNA, is preferentially incorporated into the complex that will repress target gene expression – the RNA-Induced Silencing Complex (RISC). The choice of the mature strand is based on the thermodynamic stability of the two ends of the duplex. The complementary strand (miRNA*) in most cases is degraded. However there are growing evidences that both strands can be incorporated into the RISC complex in a functional way (31).

The key proteins of the RISC complex are the Argonautes (AGO). These proteins contain three highly conserved domains, PAZ, MID and PIWI domains, that interact with the miRNAs. In mammals there are four AGO that function in miRNA repression (Ago1 to



Ago4). Different AGO proteins seems to have different specificity to the miRNA or siRNA pathway. Another crucial factor for miRNA-repression is the GW182 protein. This protein interact directly with AGO proteins and are thought to be the effectors of AGO. There are other proteins interacting with RISC to modulate miRNA function. This is the case of Fragile X Mental Retardation Protein (FMRP), which binds RNA molecules and might modulate translation. Also RNA Helicase RCK/p54, which is a p-body component, is thought to be essential to induce repression. Finally TRIM32 was recently seen to bind the RISC components enhancing in this way its activity. However further studies are required for a better understanding of the proteins that modulate this complex process of miRNA-mediated repression (32, 33, 34).

1.2.3 mRNA degradation

There are two mechanism for mRNA degradation, the first being when mRNAs undergo endolytic cleavage by Ago2 (37) and the second is when mRNAs undergo poly(A) removal by deadenylases (38).

1.2.4 Translational activation

Some studies indicate that miRNAs can stimulate translation under specific conditions. Two proteins are important for this ability of miRNAs to activate growth arrested mammalian cells. One is Ago2, the other one is FXR1, an RNA binding protein homologus to fragile X mental retardation protein FMR1/ FMRP. Surprisingly it was found that human Ago2 activates translation of target mRNAs on cell cycle arrest caused by serum starvation or contact inhibition, while it normally repressed translation of same target mRNAs in proliferating cells. FXR1 associates with Ago2 and helps to mediate the positive influence of miRNAs on translation (35, 36). Like translational repression, such activation requires base pairing between mRNA and seed region of miRNA.

1.3 miRNA target prediction

One of the most critical part in the study of miRNAs is the identification of the target genes they regulate. The study of the molecular mechanism implicated in target recognition, together with computational approaches were soon translated into basic principles used in the development of bioinformatic tools that could predict miRNAs targets.



The majority of animal miRNAs displays only modest base-pairing to their targets in contrast to what happens in plants, where the base pairing is perfect. Historically, miRNAs are known to regulate the 3’UTR of the target genes. This was experimentally demonstrated with the first miRNAs identified and it was also assumed as an in silico convenience that was further confirmed experimentally. But the use of these predictive algorithms has left underestimated the possibility that miRNAs might regulate other regions, such as the 5’UTR or even the coding region of the mRNAs. In fact, experiments involving artificial and natural mRNAs have shown that their 5’UTR can be targeted by miRNAs (39). Recent reports have also started to address the possibility that miRNAs can target the Open Reading Frame (ORF) of certain genes.

The interaction between miRNA and mRNA are through base pairing - most of the times imperfect base pairing. The most important region of the miRNA is the so called “seed” region. According to the seed “rule”, the interaction between miRNA and mRNA requires a contiguous and perfect (or nearly perfect) Watson-Crick base pairing of the 5’ nucleotides 2-8 of the miRNA. Another point to take into consideration when discussing the miRNA/mRNA interaction is the presence of multiple sites in the same 3’UTR. In fact this seems to lead to a more efficient mRNA repression. Some algorithms also take into account the conservation between related species of the miRNA-binding site. Finally one must consider that mRNAs have a secondary structure that might block miRNAs binding (40, 41, 42).

Giving different weight to each of these parameters, several algorithms were developed to predict miRNAs targets. The most known and robust ones are: TargetScan, PicTar, Miranda and PITA.

Although the principles used for target recognition are quite well established and accepted, and considering that different algorithms have different sensibilities, validation of the predicted targets is always required.

1.4 miRNA in skeletal muscle tissue

One of the first evidences that miRNAs might play a crucial role in adult skeletal muscle came from a study in sheep (43). The aim of the study was identifying the gene responsible for the hypertrophic phenotype of the Texel sheep. The authors found a point mutation in the 3’UTR of the myostatin gene that creates a new binding site for the miRNA-1 and miRNA-206. In these breed of animals, myostatin, a negative regulator of



muscle growth, is down-regulated by these two muscle-specific miRNAs inducing an exacerbated muscle growth.

Important evidence highlighting the essential role that miRNAs play in muscle came from loss-of-function experiments. In this case, O’Rourke et al (44), generated a muscle specific conditional knock-out of Dicer, the RNAse III enzyme required for miRNA maturation. In these mice, the expression of Cre recombinase was under the control of MyoD regulatory elements, and therefore started to be expressed from embryonic day 9.5. All Dicer skeletal muscle mutants died just after birth. They showed severe defects in skeletal muscle embryonic development that was mainly translated in muscle hypoplasia with hypertrophy of the few remaining fibers. This hypoplasia was attributed to an increase apoptosis rather than a defect in myofibers formation. These results are similar to the ones obtained by (45, 46) in which the knock-down of the muscle specific miRNA-1 in Drosophila caused arrest in embryogenesis with disorganized muscle development and aberrant expression of muscle-specific genes. Altogether these results anticipate a fundamental role of miRNAs in different aspects of skeletal muscle biology.

1.5 RNA seq and Transcriptome analysis

The transcriptome is the set of all RNA molecules, including mRNA, rRNA, tRNA, and other noncoding RNA produced in one or a population of cells. In multicellular organisms, nearly every cell contains the same genome and thus the same genes. However, not every gene is transcriptionally active in every cell. Different cells show different patterns of gene expression. These variations underlie the wide range of physical, biochemical, and developmental differences seen among various cells and tissues and may play a role in the difference between health and disease. Thus, by collecting and comparing transcriptomes of different types of cells or tissues, researchers can gain a deeper understanding of what constitutes a specific cell type and how changes in transcriptional activity may reflect or contribute to disease. RNA-seq is a new method in RNA sequencing to study mRNA expression. By considering the transcriptome, it is possible to generate a comprehensive picture of what genes are active at various stages of development. Not until a decade back the enormous complexity of human genome has been realized (47). Transcritome analysis has traditionally focused on cytoplasmic poly(A)+ RNA, which excluded non coding part of the genome (eg, tRNA, linc RNA, miRNA , siRNA) and hence a very large segment of important information remained



inadequately considered. But now that the non coding part of the genome is emerging as the most important regulatory population of the cell, advanced technique oriented towards delving deeper inside the transcriptome with better sensitivity to trace minor changes in gene expression and for discovering new transcripts.

Next generation sequencing provides the way for the much sought after need of understanding the complexity of transcriptome. With a dynamic range to detect subtle changes in expression level in a hypothesis-neutral environment, next generation sequencing helps provide an understanding of biological response to stimuli or environmental changes. The potential of RNA seq technology for studying transcriptome has been described by Wang et al (48). The application of RNA seq technology is vast from finding differential gene and transcript expression to revealing unannotated transcripts (49, 50).

1.6Outline of the thesis

In the present thesis, full transcriptome analysis of human skeletal muscle during development, through proliferation to differentiation has been presented in four chapters. Chapter 2 deals with the entire transcriptome sequence of human myoblast and myotube cells along with adult skeletal muscle tissue. The transcriptome analysis includes miRnome sequencing also. Entire list of differentially expressed genes has been presented. miRNA candidates have also been found. Genes of specific protein families with substantial expression has been shown. To understand the biological meaning of the genes, GO and pathway analysis results have been presented. Argonaute 2 immunoprecipitation has been performed to capture the genes bound to Ago2 and hence supposedly bound to the RISC complex,that gave us the probable candidate genes under miRNA repressing action.

In Chapter 3 differentially upregulated miRNA in myotubes presented in chapter 1 have been analyzed for their functional role. Genes enriched with Ago2 IP (chapter 1) were used for the analysis of target recognition of the miRNA.

Chapter 4 shows the transcriptome behaviour of myotubes differentiated under 3D culture conditions and their similarity and difference in comparison with muscle tissue and 2D culture has also been presented.



In Chapter 5 The response to mechanical stretch of differentiating muscle cells have been presented. Prompt response and delayed response of the transcriptome to mechanical stretch has been studied and presented in this chapter.


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Chapter - 2

Myoblast, myotube and skeletal

muscle transcriptome, miRnome

dynamics and functional profiling



2.1 Introduction:

Understanding the dynamics of muscle transcriptome during muscle growth and development is necessary to uncover the complex mechanisms underlying muscle development. The formation of skeletal muscle during vertebrate embryogenesis requires commitment of mesodermal precursor cells to skeletal muscle lineage, withdrawal of myoblasts from cell cycle and transcriptional activation of dozens of muscle structural genes. MyoD, myogenin, Myf5 and MRF4 act to establish myoblast identity and to control terminal differentiation. Myogenic bHLH factors interact with components of the cell cycle machinery to control withdrawal from the cell cycle and act combinatorial with other transcription factors to induce skeletal muscle transcription. Elucidation of these aspects of the myogenic program is leading to a detailed understanding of the regulatory circuits controlling muscle development. Skeletal muscle differentiation entails the coupling of muscle-specific gene expression to terminal withdrawal from the cell cycle. Several models have recently been proposed which attempt to explain how regulated expression and function of myogenic transcription factors ensures that proliferation and differentiation of skeletal muscle cells are mutually exclusive processes. Skeletal muscle is the dominant organ system in locomotion and energy metabolism. Postnatal muscle grows and adapts largely by remodelling pre-existing fibres, whereas embryonic muscle grows by the proliferation of myogenic cells.

The role of miRNA in muscle development is widely acknowledged now. Many groups of researchers have shown the important regulatory roles miRNA play during muscle development. miR-145 and 143 are known to regulate smooth muscle plasticity (7). Sun et al have shown the involvement of 24 in skeletal muscle differentiation (8). 181 has been shown to target Hox-A11 during myoblast differentiation (9). 1, miR-133 and miR-206 are skeletal muscle specific miRNA and have been extensively studied by many researchers (10, 11, 12).

To understand the complicated molecular networks working behind muscle development we need to obtain a thorough knowledge of the transcriptome first before the interactive networks are unravelled. We have attempted to do so by carrying out transcriptome sequencing at a massive scale. We performed the experiments in replica to have more reliable results.

The advent of next generation sequencing technology gives us the opportunity of capturing the full transcriptomic panorama of a cell at a given point of time. We used



NGS technology to our advantage and performed RNA seq of proliferating and differentiating primary human muscle cells along with adult human skeletal tissue. The functional significance of the genes differentially expressed from one developmental stage to next was studied by using Gene Ontology and pathway analyses. We also evaluated miR transcriptome profiles during the proliferative and differentiating stages of primary human muscle development. Since muscle cells ultimately grow into skeletal tissue we considered that it was worth to study the miRNA profile of adult human skeletal muscle tissue also, to understand how the miRNA population modulates itself from the cellular level to the tissue which is the ultimate functional form in the body. We were also interested in understanding the regulatory roles played by miRNAs. But we wanted to do it independent of any prediction algorithms as there are biases in prediction and also a huge number of false positives. So, we performed Ago2-RIP (Argonaute 2 RNA immunoprecipitation) experiments to capture the mRNA population that is associated with Ago-2 protein. Since Ago2 is a component of RISC complex which carries the miRNA and eventually chops off the mRNA or inhibits its translation, the mRNA associated with Ago2 most likely presents the population which is controlled by miRNA. We did the RNA seq of the immunoprecipitates and performed differential expression analysis in comparison with non Ago2 pull down RNA population. The Ago2 RIP was performed for myoblasts and myotubes. The mRNA population enriched with Ago2 was compared with mRNA differentially down regulated in myoblasts and myotubes. We did this to find out the population of genes repressed. The genes found in common were subjected to GO analyses and we saw interesting results from the analyses. The schematic representation of the experimental design is shown below:



Schematic representation of experimental plan. All the RNA seq, miRNA seq and Ago2 pull downs were performed in replica. Arrows in green show the comparison of genes enriched in Ago2 pull down versus genes down regulated. The genes found common from the comparison are most likely repressed by miRNA.

CHQ5B Myoblasts CHQ5B Myotubes Adult human skeletal muscle tissue

RNA seq + miRNA seq

Ago2 RIP + RNA seq of immunoprecipitates Genes upregulated in myoblasts (comp to myotubes) Genes downregulated in myoblasts (comp to myotubes) Genes down regulated in tissue (comp to myotubes) Genes downregulated in myotubes (comp to myoblasts) Genes upregulated in myotubes (comp to myoblasts) Genes up regulated in tissue (comp to myotubes) Genes enriched in Myotubes compared to non pull down myotubes Genes enriched in Myoblasts compared to non pull down myoblasts

Genes likely under miRNA regulation



2.2 Materials and methods:

2.2.1 Cell Culture (Human primary skeletal muscle cells (CHQ5B)):

CHQ5B primary human myoblasts were kindly provided by Dr. V. Mouly (URA, CNRS, Paris, France) (24). CHQ5B human myoblasts were isolated from the quadriceps of a newborn (5 days post-natal) without any sign of neuromuscular disorders and the protocols used for this work were in full agreement with the current legislation on ethical rules. This strain of cells can achieve 55-60 divisions before reaching proliferative senescence.

Growth conditions: DMEM (Gibco - Invitrogen) supplemented with 20% Fetal Bovine Serum (Gibco, Life Technologies) and 50µg/ml gentamycin.

Differentiation conditions: DMEM supplemented with 2% horse serum (GibcoBRL) and 50µg/ml gentamycin.

Differentiation of myoblasts into myotubes has to be induced by serum withdrawal without letting the myoblast culture reach confluency, as that reduces the myoblast population in the culture. Myotube formation can be observed after two days since serum withdrawal.

2.2.2 RNA extraction from cells: a Poly (A) extraction from cells:

Polyadenylated RNA was extracted directly from cells using QuickPrep micro mRNA purification kit (Amersham Biosciences). Cells were scrape collected after PBS washing, snap frozen in dry ice and stored at -80°C until use. 400µl extraction buffer (buffered aqueous solution containing guanidium thiocyanate and N-lauroyl sarcosine) was added to the pelleted cells and vortexed until homogenous suspension was achieved. This suspension was diluted with 800 µl of elution buffer (10mM Tris HCl, pH 7.5, 1mM EDTA) and mixed using the vortex. The mixture was centrifuged at 12,000g for 1 minute and the clear cellular homogenate was added to the Oligo(dT) beads (25mg/ml oligo dT cellulose suspended in buffer) pellet. Cellular homogenate and oligo (dT) beads were incubated together for 5 minutes at 70 -75 °C. This causes the denaturation of RNA and enhances the binding of poly (A) tail of RNA with the oligo dT beads. The sample was incubated at room temperature for 30 minutes with gentle agitation. Supernatant was



removed by centrifugation at 12,000g for 30 seconds. The oligo (dT) cellulose pellet was washed 4 times using high salt buffer (10mM TrisHCl, pH 7.5, 1mM EDTA, 0.5M NaCl) followed by washing with low salt buffer (10mM TrisHCl, pH 7.5, 1mM EDTA, 0.1M NaCl) for another 4 times. High salt conditions allow the annealing the poly(A) tail to the oligo d(T). The low salf buffer removes the poly(A)- RNAs (eg. tRNA and rRNA). These washings remove contaminating DNA, RNA proteins. The oligo dT beads were transferred to the microspin column (polypropylene minicolumns) and suspended in pre heated 100 -200 µl elution buffer (10mM Tris HCl, pH 7.5, 1mM EDTA) or pre heated RNase free water (Sigma) which releases the poly(A)+ RNA and the tube was centrifuged at 12,000g for 30 seconds. The eluate contained poly (A) RNA which was stored at -80°C until downstream processing. b Poly (A) enrichment from total RNA:

Poly(A) was enriched from total RNA preparations using Dynabeads mRNA direct kit which relies on the pairing between the poly(A)tail of mRNa and the oligo dT sequence linked to the surface of the beads. Dynabeads oligo (dT)25 are uniform, 2.8 µm diameter,

superparamagnetic, polystyrene beads with 25 nucleotide long chains of oligodeoxythymidine covalently attached to the bead surface via a 5’ linker group. RNA extraction was performed accoding to manufacturer’s instructions. The resultant mRNA was quantified using nanodrop and Qubit fluorometer. Quality assessment was performed with agilent bioanalyzer. Extraction of total ribonucleic acids (Total RNA):

Prior to extraction, all glassware was autoclaved to ensure sterility and inactivation of contaminating proteins such as nucleases. When possible, sterile plastic was used instead of glassware. To minimize the loss of nucleic acis, DNA Lobind molecular biology grade 1.5 ml tubes (Eppendorf) were extensively used. Total RNA was extracted from snap-frozen cells. 1ml TRIzol reagent (Invitrogen) was used to lyse cells grown per 10cm2 dish. The lysates were incubated with TRIzol reagent at room temperature for 5 minutes to allow complete dissociation of nucleoprotein complexes. TRIzol is a monophasic solution of phenol and guanidine isothiocyanate which maintains the integrity of RNA while disrupting cells and dissolving cell components. 0.2 ml chloroform was used per 1ml TRIzol reagent and tubes were shaken by hand for 15 seconds. This mixture was left for incubation for 2 – 3 minutes at room temperature which was followed by



centrifugation at 12,000g for 15 minutes at 4°C. Addition of chloroform followed by centrifugation separates the solution into aqueous phase and organic phase. RNA remains exclusively in the aqueous phase. The colorless upper aqueous phase containing the RNA is then transferred to a new 1.5 ml tube. An equal volume of 70% ethanol is added to the aqueous phase to obtain a final ethanol concentration of 35% and this mixture is mixed well by vortexing. This was followed by binding, washing and elution with the column of PureLink RNA mini kit (Life Technologies). Colum binding, washing and elution was performed according to manufacture’s instructions. RNA was extracted either in DNase, RNase free water (Sigma) or Tris-EDTA buffer (10mM TrisHCl, pH = 7.5, 1mM EDTA) and stored at -80°C until analysis. Small RNA enrichment from total RNA:

miRNA enrichment from total RNA sample was performed using PureLink miRNA isolation kit. 20 -30 µg of total RNA was used for small RNA enrichment. Total RNA was suspended in 90µl Nuclease free water (Sigma) to which 300µl binding buffer (L3) and 210µl 100% ethanol were added. This mixture was mixed by vortexing and then loaded onto a spin cartridge. The spin cartridge was kept in a collection tube and spun at 12,000g for 1 minute. The flow through was collected in a fresh 1.5ml tube. 700µl 100% ethanol was added to the flow through, mixed by vortexing and loaded onto another spin cartridge in a collection tube and spun at 12,000g for 1 minute. The flow through was discarded and the small RNA bound to the spin cartridge was washed twice using 500 µl wash buffer (W5) and by spinning at 12,000g for 1 minute.

Small RNA was recovered by placing the cartridge in a clean recovery tube to which 50 µl sterile, RNase free water (Sigma) was added and incubated for 1 minute. The spin cartridge was then spun at 16,000g for 1 minute and the eluted small RNA was stored at -80°C. The quality assessment of the small enriched samples was performed using Agilent small RNA chip.

2.2.3 RNA quantification and quality assessment

2.2.3a. RNA quantification:

The concentration of RNA was determined using both Qubit fluorometer (Invitrogen) and NanoDrop system (Thermo Scientific). Qubit fluorometer quantification method is based on fluorescence dyes that bind specifically to DNA, RNA or protein where as NanoDrop



quantification is based on UV absorbance measurements. Since all nucleic acids RNA, ssDNA, dsDNA absorb at 260 nm, they will contribute to the total absorbance of the sample. The degree of contamination in RNA sample preparation using NanoDrop is estimated by A260/A280 and A260/A230 ratio. A260/A280 ratio of ~2.0 is generally accepted as

“pure” for RNA. If the ratio is appreciably lower, it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm. A260/A230 ratio is a

secondary measure of nucleic acid purity. The 260/230 values for “pure” nucleic acid are often higher than the respective 260/280 values. Expected 260/230 values are commonly in the range of 2.0-2.2. If the ratio is appreciably lower than expected, it may indicate the presence of contaminants which absorb at 230 nm.

However, NanoDrop suffers a drawback. UV absorbance readings indiscriminately measure anything that absorbs at 260 nm, including DNA, RNA, protein, degraded nucleic acids, and free nucleotides. Whereas, the Qubit® Quantitation Platform, in contrast, utilizes specifically designed fluorometric technology using Molecular Probes® dyes to measure the concentration of the specific molecules of interest. These fluorescent dyes emit signals only when bound to specific target molecules, even in the presence of free nucleotides or degraded nucleic acids. Samples were prepared for RNA assay following manufacturer’s instructions. Fluorometric assay yielded quantification of nucleic acids could be compared with the data obtained using the spectrophototmeter.

2.2.3b. RNA Quality assessment:

RNA integrity which is a critical first step in obtaining meaningful gene expression data was determined using Agilent bioanalyzer and RNA Nano and Pico lab chip kits (Agilent Technologies). The Agilent Bioanalyzer is a microfluidics-based platform for sizing, quantification and quality control of DNA, RNA, proteins and cells. Profiles generated on the Agilent bioanalyzer yield information on concentration, allow a visual inspection of RNA integrity, and generate ribosomal ratios. Using electrophoretic separation on microfabricated chips, RNA samples are separated and subsequently detected via laser induced fluorescence detection. The bioanalyzer software generates an electropherogram and gel-like image and displays results such as sample concentration and the ribosomal ratio. The Agilent 2100 bioanalyzer provides a better assessment of RNA intactness by showing a detailed picture of the size distribution of RNA fragments. The RIN (RNA Integrity Number) software algorithm allows for the classification of eukaryotic total RNA, based on a numbering system from 1 to 10, with 1 being the most degraded profile



and 10 being the most intact. The labels in-between are used to indicate progressing degradation states of the RNA sample.

RNA samples were run either on RNA 6000 Nano or RNA 6000 Pico chips and Small RNA chip. Agilent small RNA chip detects RNA < 150 nucleotides long. All RNA samples were diluted according to the concentration range of respective chips. Following table gives the analytical specifications of the RNA chips.

2.2.4 Cell lysate preparation:

Cell lysate is a solution of cellular proteins resulting when cells are lysed (broken apart) under conditions which preserve the protein’s structure and function. Cell lysae preparation protocol is as follows:

The culture medium was removed and cells were washed twice with cold 1X PBS, Thorough washing is essential as insufficient washing will contaminate lysate with media components (especially serum/BSA) which may erroneously elevate protein concentration. All PBS was removed completely to prevent unwanted dilution of the final product. Cells were detached into 10 ml cold PBS with rubber policeman and the cell suspension was transferred into 15ml tube. Cells were pelleted by centrifugation at 1350 rpm for 10 minutes. Supernatant was discarded. The pellet was snap frozen in dry ice. Frozen cell pellets were used for cell lysate preparation. The frozen pellet of cells was resuspended in 300 µl lysis buffer with protease and RNase inhibitors. Resuspended pellet was homogenized by pipetting up and down, to break up the pellet thoroughly and was allowed to stand on ice for 30 minutes with intermittent vortexing every 10 minutes. The resulting mixture was centrifuged at 13,000g for 15 minutes at 4°C. This separates the debris (pellet) from total protein (supernatant). The supernatant was collected in a new 1.5 ml tube.

Protein concentration was measure using A-280 absorbance of Nanodrop (Thermoscientific).

2.2.5 Protein Analysis

2.2.5a SDS-PAGE

Protein samples were analyzed by SDS-PAGE. Resolving gels were made at 12% of a 37.5:1 mix of acrylamide/bis-acrylamide (Protogel) in 375 mM Tris HCl, pH 8.8, 0.1% v/v SDS and and 4.9 ml water. Polymerization was induced by addition of 0.1% w/v APS



and 15 µl TEMED. The separating gel was poured into a gel former. The stacking gel comprised of 4% of 37.5:1 mix of acrylamide/bis-acrylamide (Protogel) in 125mM Tris HCl pH 6.8, 0.2% v/v SDS and water. 0.1% w/v APS and 0.1% v/v TEMED were added for crosslinking. Stacking gel was poured on the top of separating gel. A gel- comb of corresponding thickness and desired number of wells was inserted in the stack gel and the assembly was left until the gel was set. Protein samples were diluted in 5X gel loading buffer and denatured at 95 °C for 5 minutes. Electrophoresis was carried out at 140V, 25mA in running buffer.

2.2.5b Western Blotting

Following electrophoresis, acrylamide gels were soaked in transfer buffer. Immobilon-P Transfer membrane (0.45 µm), two pieces of filter paper and two pieces of sponge cut to the size of the gel were also soaked in transfer buffer. Immobilon-P Transfer membrane was soaked in methanol and water briefly before being left in transfer buffer. The PDVF membrane was placed on top of the pre-soaked gel and housed between two pieces of filter paper and sponge in the western blotting cassette of Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad). The assembly was secured in a transfer tank (Mini-Protean III, BioRad) filled with transfer buffer, ensuring the gel-side of the assembly was orientated towards the cathode (and thus ensuring that proteins were transferred from the gel to the PDVF membrane as the current passed through the apparatus). Proteins were transferred to PDVF membrane at a current of 100mA for O/N. A magnetic stir-bar was placed into the tank and the whole apparatus placed on a magnetic stirrer at moderate speed to prevent the precipitation of glycine from the transfer buffer. Precipitated glycine is known to adhere to the nitrocellulose membrane, increasing background signal.

Electroblotted PDVF membrane was blocked for 3 hours at RT in Bloking buffer (Millipore) with gentle shaking. Membrane was incubated with Anti AGO2 antibody (primary antibody) diluted 1:2500 in Bloking buffer (Millipore) for 3 hours with gentle mixing. The membrane was first washed with TBS twice for 5 minutes each wash and then with TBS-T for a total of 30 minutes, changing solution every 5 minutes. Following washing, membrane was incubated with horseradish peroxidise conjugated anti Rat antibody diluted 1:1000 in bloking buffer (Millipore) for 1 hour with gentle mixing. Following 30 minutes of washing as mentioned before, immobilized protein-antibody complexes were visualized by chemiluminescence using ECL plus kit (GE Healthcare) following manufacturer’s instructions. The membrane was exposed to photographic film